Electrophoresis instruments and particularly gel electrophoresis instruments are commercially available. Conventionally, these instruments operate to move molecules of a specimen, typically DNA, RNA, proteins or peptides, in a polyacrylamide or agarose gel via electrical forces. This gel acts as a “molecular sieve” separating molecules by mass and charge. An electrical field, generated by electrodes placed on either side of the gel, forces molecules to drift through the viscous gel matrix with a drift velocity largely determined by the molecular weight of a class of molecules, the strength of the field, size and shape of the molecules, relative hydrophobicity of the sample and the ionic strength and temperature of the specimen buffer solution. Over time in a typical gel, molecules distribute in band patterns which can be subsequently stained for visualization. Using this technology, it is possible to separate and identify protein molecules that differ by as little as 1% of their molecular weight when run carefully in a gradient gel for example. This well known technique has become one of the staple tools used in molecular biology for genetic manipulation and study, DNA and protein purification, SDS PAGE, DNA footprint analysis, molecular binding studies, etc.
U.S. Pat. No. 4,558,012 to Nygren, et al issued Dec. 10, 1985, discloses the use of an ellipsometry apparatus for sensing the distribution of biomaterial in an electrophoresis operation. The Nygren patent discloses a technique in which light is reflected from the top surface of a substrate over which a gel layer is formed, the substrate having an opaque bottom surface, the light exhibiting a change in reflection intensity due to the presence of the biomaterial. The present invention differs in several respects which are necessary for more sensitive detection of thousands of molecules simultaneously. The first of these is the use of total internal reflection, described below, which monitors molecular transport within close proximity of the transparent surface. The second is the ability to image large regions of the surface in real time which is important for analyzing reaction kinetics on thousands of microarray spots for example.
U.S. Pat. Nos. 6,594,011 and 6,859,280, issued to Kempen on Jul. 15, 2003 and Feb. 22, 2005 respectively, the entire contents of which are incorporated by reference herein, disclose optical equipment for imaging binding events between patterns of probes immobilized on a surface of an optically transparent substrate and analytes to which those probes are exposed. The optical system is operative to direct polarized light to the underside of a substrate in a manner to generate an evanescent field in the plane of the probes. The equipment obtains an image of the localized changes in light intensity that occur when a probe and an analyte combine.
Most surface sensitive techniques, such as ellipsometry, cantilever detection methods, surface plasmon resonance and quartz crystal microbalance, measure the total mass of molecules within a specified region on a surface. It is therefore, very difficult to distinguish regions of the surface where multiple molecular species, often of varying mass, may adsorb. This ultimately restricts lower limits of detection of particular molecules in solution due to the presence of non-specific interactions, and complicates analysis of specific competitive reactions. This has become one of the biggest problems facing researchers working with label-free technologies.
In accordance with the present invention, the optical imaging system described in U.S. Pat. Nos. 6,594,011 and 6,859,280 and gel electrophoresis are combined in an apparatus that can measure not only the amount of biomaterial distributed over a detector slide but also the molecular weight of the molecules so distributed. In contrast to typical optical systems used, for example, in the above referenced patent to Nygren, the apparatus of the invention is not based on light absorption and does not require that the underside of the substrate be opaque. As such, the invention operates with a specimen placed in the evanescent field of a reflected light beam to provide improved imaging resolution and signal strength.